Indirect label assay device for detecting small molecules and method of use thereof

ABSTRACT

A chromatographic strip positive readout binding assay device and method suitable for quick, sensitive and reliable field testing for small molecules such as environmental contaminants, drugs of abuse, therapeutic drugs and hormones. The detectable label is not attached to either the analyte or to the analyte receptor. Low affinity binding pairs can be used in the positive readout binding assay.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 08/606,385,filed Feb. 23, 1996, issued as U.S. Pat. No. 5,874,216 on Feb. 23, 1999.

FIELD OF THE INVENTION

The present invention relates generally to the field of binding assaydevices and methods, and more specifically to a binding assay device andmethod for detecting and quantifying analyte, particularly smallmolecular weight molecules.

BACKGROUND OF THE INVENTION

The field of immunochemistry, and the development of immunoassaytechnology, has been evolving since the late 19th century. However, themajority of these methods have been developed for use by the medicalcommunity. Immunoassays are based on the highly specific binding betweenand antibody and the antigen recognized by the antibody. Antibodies arebinding proteins that are produced by the immune system of vertebratesin response to substances that are perceived to be foreign.

Various approaches have been described for carrying out immunoassays.The early ELISA's were what is commonly called a “competitive” assay inwhich the enzyme labeled antigen or antibody competed with the antigenor antibody to be determined for a reaction site on a bead, pad orsurface to which one member of an immunologically coupling pair wasattached. Subsequently, the “sandwich” assay was developed. In thesandwich assay, the antibody or antigen to be determined was“sandwiched” by an immunochemical reaction between a solid surfacetreated with an immunological species reactive with the species to bedetermined and the same or a different reactive immunological specieswhich has been coupled to a signal generating label.

Immunoassay methods combine the specific binding characteristics of anantibody molecule with a read-out system that is used to detect andquantify compounds. Immunochemical assays are reliable when used in thescreening of soil for contamination and have been used commercially forthe rapid analysis of a variety of compounds, and have been developed todetect a number of different compounds of environmental concern.

In the immunology field the term “hapten” refers to compounds that areunable to directly stimulate antibody production when injected into ananimal, but are capable of binding to antibodies if they are produced byan alternate means. Many small molecules do not stimulate the immunesystem to produce antibodies. Antibodies can be raised, however, thatspecifically bind to such small molecule haptens. For example, manyenvironmental contaminants, although potentially toxic to humans andanimals, do not elicit a strong antibody response.

A “binding assay” is an assay for at least one analyte which may bedetected by the formation of a complex between the analyte and ananalyte receptor capable of specific interaction with that analyte. Theanalyte may be haptens, hormones, peptides, proteins, deoxyribonucleicacid (DNA), ribonucleic acids (RNA), metabolites of the aforementionedmaterials and other substances of either natural or synthetic originwhich may be of diagnostic interest and have a specific ligand receptortherefor. Binding assays are generally useful for the in vitrodetermination of the presence and concentration of analyte in bodyfluids, food products, animal fluids, and environmental samples. Forexample., the determination of specific hormones, peptides, proteins,therapeutic drugs, and toxic drugs in human blood or urine hassignificantly improved the medical diagnosis of the human condition.

Current binding assay technology benefits from the diversity ofdetection systems developed that use enzyme-catalyzed chromogenicreactions, radionuclides, chemiluminescence, bioluminescence,fluorescence, fluorescence polarization, a variety of potentiometric andoptical biosensor techniques and visual labels such as latex beads, goldparticles and carbon black.

There is a continuing need for simple, rapid assays for the qualitative,semi-quantitative, and quantitative determination of such analytes in asample. Furthermore, in many situations, such assays need to be simpleenough to be performed and interpreted by non-technical users.

Binding assays rely on the binding of analyte by analyte receptors todetermine the concentrations of analyte in a sample. Analyte-receptorassays can be described as either competitive or non-competitive.Non-competitive assays generally utilize analyte receptors insubstantial excess over the concentration of analyte to be determined inthe assay. Sandwich assays, in which the analyte is detected by bindingto two analyte receptors, one analyte receptor labeled to permitdetection and a second analyte receptor, frequently bound to a solidphase, to facilitate separation from unbound reagents, such as unboundlabeled first analyte receptor, are examples of non-competitive assays.

Competitive assays generally involve a sample suspected of containinganalyte, an analyte-analogue conjugate, and the competition of thesespecies for a limited number of binding sites provided by the analytereceptor. Competitive assays can be further described as being eitherhomogeneous or heterogeneous. In homogeneous assays all of the reactantsparticipating in the competition are mixed together and the quantity ofanalyte is determined by its effect on the extent of binding betweenanalyte receptor and analyte-conjugate or analyte analogue-conjugate.The signal observed is modulated by the extent of this binding and canbe related to the amount of analyte in the sample. U.S. Pat. No.3,817,837 describes such a homogeneous, competitive assay in which theanalyte analogue conjugate is a analyte analogue-enzyme conjugate andthe analyte receptor, in this case an antibody, is capable of binding toeither the analyte or the analyte analogue. The binding of the antibodyto the analyte analogue-enzyme conjugate decreases the activity of theenzyme relative to the activity observed when the enzyme is in theunbound state. Due to competition between unbound analyte and analyteanalogue-enzyme conjugate for analyte-receptor binding sites, as theanalyte concentration increases the amount of unbound analyteanalogue-enzyme conjugate increases and thereby increases the observedsignal. The product of the enzyme reaction may then be measuredkinetically using a spectrophotometer.

Heterogeneous, competitive assays require a separation of analyteanalogue conjugate bound to analyte receptor from the free analyteanalogue conjugate and measurements of either the bound or the freefractions. Separation of the bound from the free may be accomplished byremoval of the analyte receptor and anything bound to it from the freeanalyte analogue conjugate by immobilization of the analyte receptor ona solid phase or precipitation. The amount of the analyte analogueconjugate in the bound or the free fraction can then be determined andrelated to the concentration of the analyte in the sample. Normally thebound fraction is in a convenient form, for example, on a solid phase,so that it can be washed, if necessary, to remove remaining unboundanalyte analogue conjugate and the measurement of the bound analyteanalogue conjugate or related products is facilitated. The free fractionis normally in a liquid form that is generally inconvenient formeasurements. If multiple analytes are being determined in a singleassay, the determination of the free fraction of analyte analogueconjugate for each analyte is made impossible if all are mixed in asingle liquid unless the responses of the individual analyte analogueconjugates can be distinguished in some manner. However, detecting thefree fraction of analyte analogue conjugate in assays that are visuallyinterpreted is a distinct advantage because the density of the colordeveloped in such assays is generally proportional to the analyteconcentration over much of the range of analyte concentration.

One method that can be used to detect the free analyte analogueconjugate in a heterogeneous, competitive analyte-receptor assay processis to provide a second, immobilized receptor specific for the analyte ona solid phase so that the analyte analogue conjugate not bound to thefirst analyte receptor can be bound to the second analyte receptorimmobilized on the solid phase.

A serious problem with this approach is that the concentration ofanalyte in the sample may be several orders of magnitude larger than theconcentration of analyte analogue conjugate used in the assay process.Under these circumstances, the analyte and the analyte analogueconjugate compete for the available binding sites on the first analytereceptor resulting in essentially all of the analyte analogue conjugatebeing free in the assay fluid. When the assay fluid is contacted withthe immobilized second receptor, the free analyte and the free analyteanalogue conjugate compete for binding sites provided by the secondanalyte receptor. The excess of free analyte is such that itsconcentration remains several orders of magnitude larger than that ofthe free analyte analogue conjugate so that the second analyte receptorbinding sites on the solid phase are substantially filled by theanalyte. The result of this assay process is that little or no signalmay be observed on the solid phase when the concentration of the analytein the sample is high when in fact the assay should be designed toproduce the maximum response for such concentrations of analyte.

In European Patent Application No. 87309724.0, a method is describedwhere the sample suspected of containing the analyte and a analyteanalogue conjugate are contacted with a bibulous strip that containsimmobilized analyte receptor. When sufficient analyte is present in thesample, free analyte analogue conjugate travels beyond the firstimmobilized analyte receptor zone and contacts a situs where eitheranalyte receptor or another receptor capable of binding the analyteanalogue conjugate is immobilized. If the receptor at the situs isreceptor for the analyte, then the problem of competition in thepresence of high concentrations of analyte exists as described above.Methods are described where the receptor at the situs is a receptor thatbinds to a species other than the analyte analogue on the free analyteanalogue conjugate so that high concentrations of free analyte do notcompete for binding sites at the situs. The use of such receptors at thesitus requires the development of additional analyte-receptor pairs foranalytes unrelated to the analyte for each analyte to be assayed andrestricts these assays to formats where the analyte receptor isimmobilized on a solid phase. Under these circumstances the assay ofmultiple analytes in a single assay becomes complex and difficult todevelop.

The method described in U.S. Pat. No. 4,506,009 utilizes an analyteanalogue conjugate which has both the analyte analogue and aninsolubilizing binding component coupled to the signal developmentelement. An insolubilizing receptor is used to precipitate the freeanalyte analogue conjugate unless it is sterically hindered by thebinding of the antibody specific for analyte to the analyte analogue.This method overcomes some of the deficiencies of the prior art becauseit provides a method to determine the free fraction of analyte analogueconjugate without interference from the free analyte, but it requiresthe coupling of two elements, the analyte analogue and theinsolubilizing binding component, to the signal development element insuch a way that the binding of the antibody to the analyte analoguesterically prevents the binding of the insolubilizing receptor to theinsolubilizing binding component. The relative and absolute amounts ofthe analyte analogue and the insolubilizing binding component that arecoupled to the signal development element must be empirically selectedto achieve the desired result. The need for such manipulation is bothtime consuming and may limit the assay performance by restricting theratio of analyte analogue per signal development element.

U.S. Pat. Nos. 4,094,647, 4,235,601 and 4,361,537, describe a test stripfor determining a characteristic of a sample comprising a length ofmaterial capable of transporting a developing liquid by capillary actionand having zones between its ends for receiving the sample and holdingreagents. The strip is used for performing binding assays, particularlythose in which a radioisotope is used as a label, such asradioimmunoassays. Minute sample sizes may be used. The strip is capableof application to analytical methods having sensitivities below 0.1mg/ml.

U.S. Pat. No. 3,893,808, describes a strip of filter paper treated inbands with a chemical reagent, iodine, into which a sample of gasolinesuspected of containing lead is wicked from one end and a developingreagent, dithizone, is wicked into the pretreated bands.

U.S. Pat. No. 3,895,914, describes another chemically treated test stripin which chemical reagents are applied in bands or zones on a strip fordetecting barbituric acid.

U.S. Pat. No. 4,415,700, describes hydrophilic latex beads consisting ofa homo- or co-polymer of monomers containing at least one epoxy groupand at least one polymerizable carbon-carbon double bond in eachmolecule. The method using the particles is a competitive assay whereinlabeled first antibody bound analyte and unlabeled first antibody boundanalyte compete for binding sites on a particle bound second (anti-firstantibody) antibody.

Environmental applications have been explored for the better part of adecade and a number of immunoassay methods have been developed. Mosthave been used for the detection of herbicides and pesticides in aqueousmatrixes. The application of binding assay technology to the testing ofsolid waste, complex matrixes, and highly lipophilic compounds, hasprovided unique challenges for the chemist. The feasibility ofdeveloping such methods, however, has been demonstrated withimmunoassays for single compounds such as Dioxin (see, for example,Vanderlaan, et al. Environmental Toxicology and Chemistry, 7:859-870,1988; and Stanker, et al., Toxicology, 45: 229-243, 1987).

One of the most serious problems in environmental contamination is thepresence of polychlorinated biphenyls (PCBs). PCBs, as commerciallyavailable, existed as mixtures of PCB congeners containing variousmixtures of 209 different isomeric forms. Toxicological data hasindicated that the highly chlorinated PCBs are the most toxic to humanhealth. Because the composition of PCB products varies from individualproduct to individual product, and even from lot to lot within the sameproduct, binding assay test development is difficult.

Testing is an essential and integral component of all environmentalprotection and restoration activities. It is the rate limiting elementthat influences the time, cost, and overall efficiency of projectmanagement.

The management of toxic waste sites usually involves a progressionthrough the stages of identification, characterization, remediation andmonitoring, with testing being performed during each phase. Referencelaboratory methods can effectively identify and quantify unknowncompounds in a sample, but become relatively inefficient when used torapidly locate contamination (i.e., mapping), and assist in remediationand monitoring activities. The complexity of laboratory protocols, andthe proximity of the labs to the test site, however, delays theavailability of information and increases the cost of obtaining data.The ultimate cost is in the time required by the field crews to collectand test samples for the presence of contaminants. Effective fieldscreening methods can increase the efficiency of the clean-up process byproviding an on-site, high-throughput, and cost-effective way to locatecontamination and manage its remediation.

The Environmental Protection Agency (EPA) has long promoted andsupported the concept of screening methods to supplement laboratoryanalysis and increase overall efficiency. The need for more effectivemethods has been recognized in the Superfund Amendments andReauthorization Act of 1986 which specifies the development andevaluation of alternative time and cost-saving methods that will assistin the eventual remediation of the nations Superfund sites.

Effective field screening methods can increase the efficiency of sitemanagement and improve overall data quality when used to supplement theservices of regional laboratories. The development of these methods,however, requires a technology that will be compatible with numerouscompounds and matrixes and yet be simple, effective and rugged enough tobe incorporated into a protocol for use in the field.

Screening methods need to provide fast, simple, cost-effective andreliable information when operated under field conditions. The reagentsand equipment should be portable and stable at ambient conditions, andthe claims relating to performance should accurately reflect anticipatedfield use. The methods should be able to rapidly provide an amplequantity of data, and the protocol should be simple to perform and safeto use. Performance characteristics relative to sensitivity, freedom,and correlation to an acceptable reference method should be carefullyevaluated. A necessary characteristic of particular significance forscreening methods is that they exhibit a very low frequency of falsenegative results.

Screening methods detect contamination at specified concentrations. Theconcentration may relate to a hazardous threshold, a clean-up target, ora process-control parameter. The potential implications of falsenegative data far outweigh those of false positive results. Theconsequence of a false positive, while a costly problem that needs to beminimized, results in additional testing or treatment. False negativedata, however, provides an erroneous perception of a clean site, and mayhave serious environmental and legal consequences. Safeguards thatminimize the incidence of false negative results are imperative.

Thus, a simple binding assay method is needed which will providereliable, accurate and fast results in the field for a wide range of PCBcontaminants (regardless of manufacturer, exact composition or matrix)in a single test using a single antibody. Such an assay would increasethe efficiency of environmental site management activities such ascharacterization (mapping), remediation monitoring, and regulatorycompliance.

Similarly, the most widely used products responsible for environmentalcontamination are refined petroleum products. The contamination of soiland ground water by petroleum products during transport, storage,treatment and disposal is a frequent occurrence. In an attempt toestablish the magnitude of the problem, a recent study by the EPA'sUnderground Storage Tank Program estimated that the U.S. containsapproximately 1.4 million underground storage tanks and as many as400,000 of these tanks may be leaking (see Schwenndeman, et al, inUnderground Storage Systems: Leak Detection and Monitoring. LewisPublishers, Inc., Chelsea, Mich., 1987, 16; Federal Register, Vol. 52,No. 74, 12664 (1987)).

Currently, reference methods for detecting contamination at sitesinclude analysis for benzene, toluene, xylene, ethyl benzene andpetroleum hydrocarbons. These methods require laboratory analysis by gaschromatography (GC) or infrared (IR) methods and an extended period oftime to obtain the results (see Potter, in Petroleum Contaminated Soils,eds., Calabrese et al., Vol. 2, Lewis Publishers, Chelsea, Mich., 97(1990)).

While hydrocarbon vapor analyzers can be useful because they providerapid results, they fail to accurately reflect the amount ofhydrocarbons in a soil sample and are unable to detect the morepersistent contamination caused by semi-volatile components.

Thus, a binding assay method is needed which will provide reliableaccurate and fast results in the field for a wide range of petroleumbased contaminants, regardless of manufacturer or exact composition.Such an assay would increase the efficiency of environmental sitemanagement activities such as characterization (mapping), remediation,monitoring, and regulatory compliance.

A major disadvantage of prior art positive readout competitive bindingassay devices and methods used for detecting small molecules is that thebinding affinity of the analyte and its associated binding partner mustbe sufficiently high to prevent false positives. However, many smallmolecule binding pairs have insufficient affinity, thus limiting theapplicability of this technology to the detection and measurement ofsmall molecules critical for the accurate assessment of environmentalquality.

There is also a great need for simple, inexpensive and easy-to-usedevices that can be used in doctor offices, at clinics or at home fortesting for the presence of therapeutic drugs or hormones in bodyfluids. Additionally, clinics, emergency medical technicians,corrections facilities, police and firemen require an affordable andeasy-to-use device suitable for quickly testing for the presence ofdrugs of abuse in body fluids outside of a hospital setting.

Thus, a simple, inexpensive, reliable binding assay device and methodthat is rapid, accurate and precise, and capable of detecting andmeasuring small molecules and adaptable for field testing is needed.

SUMMARY OF THE INVENTION

The present invention relates to a binding device and assay method thatovercome the problems of the prior art.

It is an object of the invention to provide a simple, inexpensive,reliable binding assay device and method that is rapid, accurate andprecise.

It is a further object of the invention to provide a binding assay andmethod useful for detecting small molecules.

A feature of the invention is that the label is an indirect label, i.e.the label to be detected is not conjugated to either the analyte or tothe analyte receptor, but rather is conjugated to a binding moietycapable of specifically binding to either the analyte carrier conjugateto be detected or to a diffusable analyte receptor. The labeled bindingmoiety-conjugate is diffusable and is provided in a limited quantityrelative to the amount of label binding moiety binding partner presentin the device.

Another feature of the invention is that the binding assay deviceprovides a positive readout signal.

Another feature of the invention is that the device and method aresuited for rapid and reproducible analyses.

An additional feature of the invention is that the diffusable bindingpartner of the binding pair of the analyte to be detected is theanalyte, then the analyte is conjugated to a carrier that is capable ofbeing bound by an immobilized binding moiety.

An advantage of the invention is that binding partners with bindingaffinities of less than approximately 10⁻⁷, which would not otherwise beappropriate for a positive readout competitive binding assay, can beused in a lateral flow binding device to achieve accurate andreproducible results.

Another advantage is that the binding assay device and method results ina signal that increases in proportion to the concentration of analyte inthe test sample, providing an easy to visualize positive signal. Incontrast, standard competitive binding assays provide a signal that isinversely related to the amount of analyte in the sample, which isdifficult to assess visually.

An advantage of the limiting quantity of indirect label used in thebinding assay device and method of the invention is that “bleed-through”and false positives are substantially reduced or eliminated.

Yet another advantage is that the device and method are particularlysuited for field testing and screening of samples for the presence ofsmall molecules, without the need for extensive training or expensivelaboratory equipment.

The invention more specifically relates to a chromatographic stripindirect label positive readout competitive binding assay devicecomprising,

a chromatographic strip having a length and narrow width capable ofconveying fluids in a fluid flow direction generally parallel to thelength of the strip, the strip comprising the following zones:

(1) a sample contact zone where fluid sample suspected of containing ananalyte may be contacted with the strip;

(2) a labeled anti-first binding partner zone disposed at, or downstreamfrom the sample contact zone comprising diffusable labeled anti-firstbinding partner;

(3) a first binding partner zone disposed at, or downstream from thelabeled anti-first binding partner zone comprising diffusable unlabeledfirst binding partner in an amount sufficient to bind approximately atleast all of the labeled anti-first binding partner;

(4) a trapping zone on the strip at, or downstream from the firstbinding partner zone comprising immobilized second binding partner in anamount sufficient to bind at least a sufficient amount of the unlabeledfirst binding partner to bind substantially all of the labeledanti-first binding partner; and

(5) a detection zone on the strip at a downstream location from thetrapping zone comprising an immobilized binding moiety specific for thefirst binding partner.

When the first binding partner is analyte, the first binding partner isprovides as an analyte-carrier conjugate, the binding moiety desirablyspecifically binds the carrier portion of the conjugate, and the secondbinding partner is analyte receptor. When the first binding partner isanalyte receptor, the second binding partner is analyte-carrierconjugate.

The device and method of the present invention permit rapid, low costfield testing and screening for important small molecules, such asenvironmental contaminants, drugs of abuse, therapeutic drugs andhormones.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of a binding assay device.

DETAILED DESCRIPTION OF THE INVENTION

This invention permits the utilization of a low affinity binding partnerfor the analyte, yet avoids the problem of false positive signals commonto positive readout binding assays using low affinity binding partners.The invention utilizes:

(1) a diffusable unlabeled first binding partner, which generally haslow binding affinity for a second binding partner. When the firstbinding partner is analyte, the first binding partner is provided asanalyte-carrier conjugate, wherein the carrier portion of the conjugateis capable of being bound by an immobilized binding moiety;

(2) a diffusable labeled anti-first binding partner, which usually hashigh binding affinity for the first binding partner;

(3) an immobilized second binding partner, which generally has lowbinding affinity for the first binding partner. When the second bindingpartner is analyte, the second binding partner is provided asanalyte-carrier conjugate; and

(4) an immobilized detection binding moiety capable of binding to thefirst binding partner.

The diffusable unlabeled first binding partner is provided in an amountsufficient to bind approximately all of the diffusable labeledanti-first binding partner, and the immobilized second binding partneris provided in an amount sufficient to bind a sufficient amount of thediffusable unlabeled first binding partner to bind approximately all ofthe diffusable labeled anti-binding partner.

In the absence of analyte in a sample applied to the binding device, allof the label is trapped prior to the detection zone, yielding a negativeresult. The presence of analyte in the sample produces a positivereadout signal that increases proportionately as the amount of analytein the sample increases.

Important terms used herein are defined as follows.

The term “analyte” refers to a chemical, toxin or compound to bemeasured by the device and method of the invention. The analyte is abinding partner for the analyte recepter. Generally, the analyte is asmall molecule having a molecular weight of less than approximately10,000 Daltons, desirably less than 5,000 Daltons, more desirably lessthan 2,000 Daltons, still more desirable less than 1,000 Daltons. Theanalyte may be, for example, an antigen or a ligand, or a fragment ofRNA or DNA. Generally, the analyte is conjugated to another molecule,herein referred to as a carrier, thus providing a diffusable unlabeledanalyte-carrier conjugate. Suitable carrier molecules include proteins,polymer particles, carbohydrate polymers, liposomes and beads.

The term “analyte receptor” refers to a molecule capable of bindingspecifically with the analyte. The analyte receptor may be a protein,peptide, fragment of RNA or DNA, binding moiety or otherwise a bindingpartner of the analyte. A novel aspect of the invention is that thepositive readout binding assay device and the method of testing foranalyte may employ low affinity binding partners, for example bindingpartners having binding constants of less than approximately 10⁻⁷,desirably approximately 10⁻⁵ to 10⁻⁷.

The analyte and the analyte receptor together are referred to as abinding pair comprising a first binding partner and a second bindingpartner. The analyte and the analyte receptor may be either the firstbinding partner or the second binding partner, respectively, dependingon the particular embodiment of the invention. The analyte is generallyprovided in the binding device as analyte-carrier conjugate. Desirablebinding pairs include antibody-antigen, antibody-hapten; andreceptor-ligand binding pairs such as aryl hydrocarbon receptor—arylhydrocarbon (e.g. dioxin, polyaromatic hydrocarbon and furan); hormonereceptor hormone (e.g. estrogen); nucleic acid hybridizing oligomersDNA/RNA fragments (e.g. from Cryptosporidium or Giardia); artificialbinding partners (e.g. molecular imprinting—the process of makingmolecule casts of small molecules by combining a resin with the smallmolecule, crosslinking or otherwise hardening the resin, grinding theresin and extracting the small molecule resulting in smallmolecule-specific casts capable of specifically binding the smallmolecule, and RNA nexamers—functionalized RNA oligomers capable ofacting as a specific binding partner).

Conjugation of the analyte to form a diffusable binding partner-carrierconjugate is carried out using standard conjugation techniques wellknown in the art. Suitable cross-linking reagents in accordance with thepresent invention include, but are not limited to the following:S-acetylthioglycolic acid, N-hydroxysuccinimide ester;N-(5-azido-2-nitrobenzyloxy) succinimide; p-azidophenacyl bromide;N-(4-azidophenylthio) phthalimide; 4-azidosalicylic acidN-hydroxysuccinimide ester; bromoacetic acid N-hydroxysuccinimide ester;1,4-butanediol diglycidyl ether; 2-diazo-3,3,3-trifluoropropionic acidp-nitrophenyl ester; dimethyl adipimidate dihydrochloride; dimethyl3,3-dithiobispropionimidate dihydrochloride; dimethyl pimelimidatedihydrochloride; dithiobis(propionic acid N-hydroxysuccinimide ester);ethylene glycol bis-(succinic acid N-hydroxysuccinimide ester);4-fluoro-3-nitrophenyl azide; bis(4-fluoro-3-nitrophenyl) sulfone;4-(N-maleimido)benzophenone; g-maleimidobutyric acidN-hydroxysuccinimide ester; e-maleimidocaproic acid N-hydroxysuccinimideester; 4-(N-maleimidomethyl)cyclohexane-1 carboxylic acidN-hydroxysuccinimide ester; β-maleimidopropionic acidN-hydroxysuccinimide ester; N,N-bis(3-maleimidopropionyl)-2 hydroxy-1,3propanediamine; N,N-o-phenylene dimaleimide; polyoxyethylenebis(glycidyl ether); polyoxyethylene bis(imidazolyl carbonyl) andsuberic acid bis(N-hydroxysuccinimide) ester, etc.

Moreover, suitable linker molecules in accordance with the presentinvention include, but are not limited to, the following: proteins,carbohydrates, lipids, peptides, polyesters, nucleic acids and syntheticpolymers (e.g., poly-1-lysine) (See. e.g. Auotsuka, S., et al., J.Immunol. Methods., 28:149-162 (1979); Presentini, R., et al., J.Immunol. Methods, 10:395-412 (1989) and Vershcoor, J. A. et al., J.Immunol. Methods, 127: 43-49 (1990), the disclosure of which areincorporated herein by reference). The linker molecule can bebifunctional (i.e., it may have two reactive sites) wherein one of thereactive sites is used for attachment to the analyte, while the otherreactive site is used to attach the analyte conjugate partner.Generally, the analyte can be attached to the linker molecule and, inturn, the carrier using a cross-linking reagent, supra, in a Schiff baseformation reaction with an aldehyde group; an amide formation reactionwith an amine or carboxylic acid group using a peptide coupling reagent(such as, for example, carbodiimide, acid chloride and the like, supra);an ester formation reaction with a hydroxyl or carboxylic acid groupusing condensing agents; a sulfide formation reaction using a sulfidecoupling agent, supra; an azo bond formation reaction with an aromaticdiazonium salt; or other known coupling reactions for joining organicmolecules. See, reviews of such methods and techniques: e.g., Kabat, E.A. Structural Concepts In Immunology and Immunochemistry (2nd Ed., Holt,Rinchart and Winston, New York (1976); Eyzaguirre, J. ChemicalModifications of Enzymes; Active Site Studies(John Wiley andSons(1982)); Wong, S. S., Chemistry of Protein Conjugation andCross-Linking (CRC Press, Inc. Boca Ration, 1991); and Brinkley, M.Bioconjugate Chem. 3:2-13 (1992), the disclosures of which areincorporated herein by reference.

The term “label” refers to any suitable label capable of being attachedor conjugated to a anti-first binding partner, and being detected andquantified. Examples of suitable labels include enzymes capable ofreacting to produce a colored reaction product, such as horseradishperoxidase and alkaline phosphatase, molecules capable of producingdetectable light such as bioluminescence, chemiluminescence,phosphorescence and fluorescence, and particles, such as carbon black,colored latex beads or gold particles.

The term “chromatographic strip” refers to any substrate upon whichtrapping zone molecules and detection zone molecules can be immobilized,and which is capable of conveying fluid in a fluid flow direction thatis generally parallel to the length of the chromatographic strip.Desirable chromatographic strips comprise non-woven substrates,including without limitation nylon, polyethylene, glass fiber, andcellulose. A particularly desirable chromatographic strip isnitrocellulose. The substrate of the chromatographic strip optionallymay be backed with, or laminated to, another material. Desirable backingor laminating material is polyethylene or vinyl, although other suitablematerials known in the art may be used.

The term “sample contact zone” is the region, generally located at oneend of the chromatographic strip, where sample is applied to the stripand from which sample fluid flows in a fluid flow direction towards theopposing, or distal, end of the strip. The sample contact zone mayoptionally comprise a filter material. Examples of filter materialinclude filter paper, glass fiber, polyethylene and other suitablefilter materials know to skilled artisans.

The term “binding moiety” refers to any molecule capable of specificallybinding to its indicated binding partner. Generally, the binding moietyhas high affinity for its binding partner, and is immobilized at thedetection zone. Examples of binding moieties include polyclonal ormonoclonal antibodies, or fragments of such antibodies containing thebinding regions, receptors such as the dioxin receptor or hormonereceptors, and fragments of RNA or DNA. Where the binding partnercomprises an antibody, the binding moiety may be a second antibody, orbinding portion thereof, specific for the first antibody, for example agoat anti-mouse antibody. Where the binding moiety is immobilized in thedetection zone, the preferred binding partner is the diffusableunlabeled first binding partner.

The term “diffusable” refers to the ability of the molecules to flow inthe fluid flow direction downstream along and throughout thechromatographic strip towards the distal end of the strip.

Having defined important terms used herein, the invention and desirableembodiments thereof will be described in detail below.

The present invention encompasses a positive readout assay device thatis designed to detect important analytes, particularly small moleculessuch as environmental and food contaminants, chemicals, toxins andcompounds, metabolites such as hormones, drugs of abuse, and therapeuticdrugs and metabolic byproducts thereof. These analytes are generallysmall molecules, which in the past have been difficult to detect andmeasure accurately and reproducibly in the field by non-technicalpersonnel using binding assays.

By positive readout is meant a competitive binding assay providing asignal that increases proportionally as the concentration of analyte inthe test sample increases. The positive readout feature of the inventionprovides an easy to determine indication of a positive result. This is asubstantial improvement over standard lateral flow competitive bindingassays.

Typically small molecules are detected and quantified using a standardcompetitive inhibition assay which results in an inverse relationshipbetween analyte concentration and signal generation. As theconcentration of the analyte in a test sample increases, the signalgenerated from the assay decreases. Thus, in standard competitivebinding assays the greatest signal generation occurs when the analyte isabsent from the test sample. Therefore, it is difficult to visuallydetermine a low concentration of analyte because the signal change isfrom a dark color to a slightly less dark color.

An improvement of the competitive assay format provided by the presentinvention is the development of a positive readout assay, e.g., an assayin which the signal increases a person's ability to visually detect lowconcentrations of analyte. The detection of a lightly colored band on awhite membrane is much easier to see than a slight decrease in a darkband on the membrane.

The invention also encompasses a method of testing for analytes usingthe binding assay device. A novel aspect of the invention is the abilityto use binding pairs having binding affinities of less than 10⁻⁷,desirably between 10⁻⁵ and 10⁻⁷.

Referring to FIG. 1, the binding assay device of the inventioncomprises,

a chromatographic strip 10, comprised of a substrate 11, generallyhaving a length and narrow width with a proximal end 12 and a distal end13, which is capable of conveying fluids in a fluid flow directiongenerally parallel to the length of the strip. The chromatographic stripcomprises multiple zones. A sample contact zone 15 located at theproximal end of the strip, is the region where fluid sample suspected ofcontaining an analyte may be contacted with the strip. A diffusablelabeled anti-first binding partner zone 20, is disposed downstream(towards the distal end of the strip) from the sample contact zone 15comprising diffusable labeled binding moiety capable of binding a firstbinding partner. The first binding partner is either analyte or analytereceptor. When the first binding partner is analyte, the first bindingpartner is desirably a conjugate of analyte and carrier. The labeledanti-first binding partner may specifically bind any portion of thefirst binding partner, including the carrier portion when it is aconjugate. An unlabeled first binding partner zone 30 is disposeddownstream from the anti-first binding partner zone 20. The unlabeledfirst binding partner is provided in an amount sufficient to bindapproximately all of the diffusable labeled anti-first binding partner Atrapping zone 40 is immobilized on the strip at a downstream locationfrom zones 20 and 30, comprising second binding partner in an amountsufficient to bind at least a sufficient amount of the diffusable firstbinding partner to bind substantially all of the diffusable labeledanti-first binding partner. Finally, a detection zone 50 is immobilizedon the strip at a downstream location from trapping zone 40 comprising abinding moiety specific for the first binding partner, desirably thecarrier when the first binding partner is a conjugate. The anti-firstbinding partner of zone 20 may optionally be the same as the bindingmoiety of zone 50.

For clarity each zone is depicted as being separated from every otherzone. However, one or more zones may be coincident or overlap, with theexception of the detection zone, which must always be separate anddownstream from the other zones.

In another embodiment of the invention, the labeled anti-first bindingpartner zone, or first binding partner zone, or both, are not placed onthe strip. In these embodiments, the labeled anti-first binding partner,or first binding partner, or both, are combined with the sample prior toapplication of the sample to the sample contact zone. The thus formedmixture of sample and assay components is then applied to the samplecontact zone.

One embodiment of the method entails combining both diffusable assaycomponents with the sample prior to applying the sample-assay componentmixture to the binding assay device. More particularly, the presence andamount of an analyte in a sample suspected of containing the analyte isdetected by employing a chromatographic strip-positive readout bindingassay device, the binding assay device comprising,

a chromatographic strip having a length and narrow width capable ofconveying fluids in a fluid flow direction generally parallel to thelength of the strip, the strip comprising;

a sample contact zone whereat fluid sample suspected of containing ananalyte may be contacted with the strip;

a trapping zone located at, or downstream from, the sample contact zonecomprising immobilized second binding partner in an amount sufficient tobind at least a sufficient amount of the unlabeled first binding partnerto bind substantially all of the labeled anti-first binding partner; and

a detection zone located on the strip downstream from the trapping zonecomprising an immobilized binding moiety specific for the diffusableunlabeled first binding partner,

wherein the sample to be tested is combined with diffusable labeledanti-first binding partner and diffusable unlabeled first bindingpartner to form a sample-assay component mixture, and the sample-assaycomponent mixture is applied to the sample contact zone, and thepresence of label in the detection zone is detected.

Another embodiment employs a chromatographic strip-positive readoutbinding assay device, the binding assay device comprising,

a chromatographic strip having a length and narrow width capable ofconveying fluids in a fluid flow direction generally parallel to thelength of the strip, the strip comprising;

a sample contact zone whereat fluid sample suspected of containing ananalyte may be contacted with the strip;

a labeled anti-first binding partner zone disposed at, or downstreamfrom the sample contact zone comprising diffusable labeled anti-firstbinding partner;

a trapping zone located at, or downstream from, the sample contact zonecomprising immobilized second binding partner in an amount sufficient tobind at least a sufficient amount of the unlabeled first binding partnerto bind substantially all of the labeled anti-first binding partner; and

a detection zone located on the strip downstream from the trapping zonecomprising an immobilized binding moiety specific for the diffusableunlabeled first binding partner,

wherein a sample to be tested is combined with diffusable unlabeledfirst binding partner to form a sample-assay component mixture, thesample-assay component mixture is applied to the sample contact zone,and the presence of label in the detection zone is detected.

Yet another embodiment employs a chromatographic strip-positive readoutbinding assay device, the binding assay device comprising,

a chromatographic strip having a length and narrow width capable ofconveying fluids in a fluid flow direction generally parallel to thelength of the strip, the strip comprising;

a sample contact zone whereat fluid sample suspected of containing ananalyte may be contacted with the strip;

a first binding partner zone disposed at, or downstream from the samplecontact zone comprising diffusable unlabeled first binding partner in anamount sufficient to bind approximately all of a labeled anti-firstbinding partner in a sample-assay component mixture to be applied to thebinding device;

a trapping zone located at, or downstream from, the sample contact zonecomprising immobilized second binding partner in an amount sufficient tobind at least a sufficient amount of the unlabeled first binding partnerto bind substantially all of the labeled anti-first binding partner; and

a detection zone located on the strip downstream from the trapping zonecomprising an immobilized binding moiety specific for the diffusableunlabeled first binding partner,

wherein a sample to be tested is combined with diffusable labeledanti-first binding partner to form a sample-assay component mixture, thesample-assay component mixture is applied to the sample contact zone,and the presence of label in the detection zone is detected.

The sample contact zone 15 may optionally comprise a filter materialoverlay 35 through which the sample flows to reach the sample contactzone. Additionally, absorbent material may be attached to the distal endof the chromatographic strip to facilitate the flow of sample fluid fromthe sample contact zone past the downstream zones. A cover or casinghaving openings for the sample contact zone and the detection zone mayoverlay or surround the chromatographic strip. The cover or casingdesirably is made of non-absorbent material such as plastic.

The cover or casing optionally may be formed so as to provide avolumetric container into which the sample is introduced. The containermay be either integral with the cover or casing, or be separate from thecover or casing, but attachable to the cover or casing at the samplecontact zone opening so as to form a volumetric cavity into which sampleis introduced. The sample may be poured or otherwise introduced into thevolumetric container to a predetermined volume, for example to the topof the container, or to a line scribed, printed or embossed on the sideof container. Alternatively, the container may contain an opening suchas a slot or hole such that when the container is submerged in sampleand withdrawn, excess sample volume runs out of the container.

It is believed that because there is an excess unlabeled first bindingpartner and of immobilized second binding partner, relative to theconcentration of labeled anti-first binding partner present in thedevice, the labeled anti-first binding partner is essentially completelybound by the first binding partner which, itself, is bound to theimmobilized second binding partner, thereby preventing bleed-through andfalse positives. Even low affinity binding partners, having a bindingaffinity of less than approximately 10⁻⁷ may be used in the bindingassay of the invention.

The method of the invention comprises applying sample to be tested, or amixture of sample and assay components as described above, to the samplecontact zone and detecting the presence, and optionally the amount, oflabel captured at the detection zone. The application of sample may beby any standard application, for example via dropper, syringe, pipet orpouring, or by dipping the chromatographic strip in sample such thatsample contacts the sample contact zone.

The detection of the label may be any standard visualization, detectionor measurement means known in the art. Desirable methods for detectingand quantifying the label in tile binding assay invention includeenzymatic color generation, radioactive detection such as by X-rayexposure of films or scintillation or gamma counting, depending on theisotope utilized, luminescent measurements where the label producesbioluminescent or chemiluminescent light, and fluorescent. Desirabledetection methods are visual assessment, color charge, for example dueto pH shift, and measurement amount of colored label with a hand-heldreflectometer.

Prior to application of sample to the binding assay device, the samplemay be pre-treated to extract, concentrate or release the analyte to bedetected. For example soil samples may be methanol treated to extractcontaminants, toxins or compounds who presence and quantity are usefulas indicia of the environmental quality of the soil being tests.

Multiple analytes may be tested simultaneously according to the presentinvention. For example, multiple diffusable labeled binding partners forvarious analyte binding pairs could be applied to a binding assaydevice. The presence of label at the detection zone indicates that atleast one of the analytes for which the device is sensitive is presentin the sample. In this way rapid screening of multiple environmentalfactors can be conducted with a minimum number of test, at a reducecost, and in an expedient fashion.

Alternatively, the binding assay device of the invention may incorporatemultiple diffusable label binding partners as described above incombination with multiple detection zones, each zone being specific foran individual analyte-specific label. This device provides a means forrapid, low cost environmental screening, while retaining the abilitydiscriminate between, and quantify, various specific analytes.

In one embodiment the diffusable labeled anti-first binding partner islabeled anti-BSA antibody, the diffusable unlabeled first bindingpartner-carrier conjugate is PCB-BSA, the immobilized second bindingpartner is anti-PCB antibody, and the detection binding moiety isanti-BSA antibody.

In another embodiment the diffusable labeled anti-first binding partneris labeled goat anti-mouse antibody, the diffusable unlabeled firstbinding partner-carrier conjugate is anti-PCB antibody, the immobilizedsecond binding partner is PCB-BSA, and the detection binding moiety isanti-mouse antibody.

The invention may be further understood by reference to the followingnon-limiting detailed examples.

EXAMPLE 1 Analyte Diffusable and Analyte Receptor Immobilized

I. Membrane Coating

A. Cut membranes in 0.8 cm×5 cm strips

B. Dilute goat anti-mouse IgG to 1 mg/ml in phosphate buffered saline(PBS)

C. Add goat anti-mouse about 2.0 cm from top of strip

D. Add anti-fluorescein (1 mg/mL) about 1.0 cm from top of strip

E. Dry strips

F. Block strips with PBS/Casein (0.5%) for 30 minutes

G. Dry strips

H. Anti-PCB antibody (approx. 100 mg/mL) in PBS is applied to the goatanti-mouse IgG band

I. The strip is dried, washed and dried again

J. Add PCB-BSA-FITC (10 mg/mL) 2.5 cm from top

K. Add Anti-fluorescein gold particles in Tris/BSA buffer (Tris (20 mM),BSA (1%), sodium azide (0.1%), pH 8.2) containing 30% sucrose about 3 cmfrom top of the strip

L. Dry strip

II. Protocol for Coating Gold Particles with Rabbit Anti-fluorescein IgG

A. Adjust pH of gold to about 9. Add final concentration of 5 mM sodiumborate (Na₂B₄O₇), pH=9.2

B. Dialyze rabbit anti-fluorescein against 2 mM borate (pH 9.2). 3changes of 100×column of antibody for 24 hour. Antibody should be >2mg/mL.

C. Add 0.06 mg Ab/mL of gold—rapidly mix

D. Incubate for 2-15 min. at RT

Check spectrum between 450-800 nm

Compare gold solution (50 mL) diluted into water (450 mL) and into 10%NaCl solution (450 mL)

If spectrum do not match, then the gold particles have not absorbedenough protein and one should add another 0.03 mg Ab/mL gold. Continueadding antibody until spectrum match. The spectrum for the saltcontaining sample will be slightly lower than the water sample but itshould have the same shape.

E. Add {fraction (1/10)} volume 10% BSA in water=final of 1% BSA. Mixrapidly upon addition and incubate 2-15 minutes at RT.

F. Centrifuge for 5 min @ 10,000×g. Discard supernatant.

G. Resuspend pellet in Tris (20 mM), BSA (1%), sodium azide (0.1%), pH8.2, using ½ the original volume.

H. Centrifuge for 5 min @ 8,000×g. Discard supernatant. Resuspend pelletin Tris/BSA buffer.

I. Repeat washes two more time. The last spin may require 10,000×g.

J. Resuspend pellet in ⅕ original volume

III. Preparation of PCB-BSA-FITC

A. Dissolve BSA (Fraction V) in Potassium Phosphate (0.2 M, pH 8) at 10mg/mL.

B. Use PCB activated with the n-hydroxysuccinimidyl ester (PCB-NHS) at54 mmoles/mL.

C. Make BSA solution 33% DMF include volume of PCB-NHS (total of 0.5 mLper mL BSA)

Add 446 mL DMF per 10 mg BSA, mix

Vortex while adding 54.4 mL of PCB-NHS per 10 mg BSA (20 fold excess)

D. Incubate 1 hour at RT

E. Centrifuge for 5 min. at 14,000 rpm

F. Put the supernatant through a G-25 column (1.5 mL for each disposable10 mL column) equilibrated with PBS/0.05% azide.

G. Collect protein fraction

H. Dissolve FITC in DMF and add a 20 fold molar excess to the PCB-BSAconjugate. Incubate for 1 hour at room temperature.

I. Purify using a G-25 column as in step F.

J. Collect protein fraction

IV. Assay

A. Attach an absorbent pad to top and bottom end of strip.

B. Add several drops of sample to bottom absorbent pad of the strip andallow to “wick” up the strip.

C. Allow sample to wick up entire length of strip.

D. If color develops in the detection zone (approx. 1 cm from end ofstrip), then the sample contains PCB.

EXAMPLE 2

The binding device of Example 1, except that PCB-HRP conjugate issubstituted for the PCB-BSA conjugate. The PCB-HRP conjugate is made asfollows.

A. Dissolve 16 mg of HRP in 1 mL of distilled water

B. Add 2 mg of NaIO₄, incubate for 10 min at RT.

C. Purify the HRP on a G-25 column equilibrated with 200 mM sodiumcarbonate buffer at pH 9.

D. Add 1 mg of PCB-hydrazide at 10 mg/mL in methanol.

E. Incubate for 1 hour at room temperature.

F. Add 1.5 mg of NaBH₄, incubate for 50 min at 4° C.

G. Dialyze against 3 changes of phosphate buffered saline bufferovernight.

EXAMPLE 3 Analyte Receptor Diffusable, Analyte Immobilized

I. Membrane Coating:

A. Cut membranes in 0.8 cm×5 cm strips

B. Dilute goat anti-mouse IgG to 5 mg/ml in phosphate buffered saline(PBS)

C. Add goat anti-mouse about 1.0 cm from top of strip

D. Add PCB-BSA analyte-conjugate (10 mg/mL) about 2.0 cm from top ofstrip

E. Dry strips at 37° C. for 15 minutes

F. Block strips with PBS/Casein (0.5%) for 30 minutes

G. Dry strips at 37° C. for 1 hour

H. Add anti-PCB antibody (approx. 10 mg/mL) in PBS and 30% sucrose about2.5 cm from top of strip

I. Add goat anti-mouse gold particles in Tris/BSA buffer (Tris (20 mM),BSA (1%), sodium azide (0.1%), pH 8.2) containing 30% sucrose about 3 cmfrom the top of the strip

J. Dry strip at 37° C. for 1 hour

K. Perform assay (see Section IV)

II. Protocol for Coating Gold Particles with Goat Anti-mouse IgG

A. Adjust pH of gold particle suspension to about 9. Add finalconcentration of 5 mM sodium borate (Na₂B₄O₇), pH=9.2

B. Dialyze goat anti-mouse IgG against 2 mM borate (pH 9.2). 3 changesof 100×volume of antibody for 24 hours. Antibody concentration shouldbe >2 mg/mL

C. Add 0.06 mg Ab/mL of gold—rapidly mix

D. Incubate for 2-15 minutes at room temperature

Check spectrum between 450-800 nm

Compare gold solution (50 mL) diluted into water (450 mL) and into 10%NaCl solution (450 mL).

If spectrum do not match, then the gold particles have not absorbedenough protein and one should add another 0.03 mg Ab/mL gold. Continueadding antibody until spectrum match.

E. Add {fraction (1/10)} volume 10% BSA in water=final of 1% BSA. Mixrapidly upon addition and incubate 2-15 minutes at room temperature.

F. Centrifuge for 5 minutes at 10,000×g. Discard supernatant.

G. Resuspend pellet in Tris (20 mM), BSA (1%), sodium azide (0.1%), pH8.2, using ½ the original volume. Vortexing should be sufficient tocompletely resuspend pellet.

H. Centrifuge for 5 minutes at 8,000×g. Discard supernatant. Resuspendpellet in Tris/BSA buffer.

I. Repeat wash two more times. The last spin may require 10,000×g

J. After last spin, resuspend pellet in ⅕ original volume

III. Preparation of PCB-BSA

A. Dissolve BSA (Fraction V) in Potassium Phosphate (0.2M, pH 8) at 10mg/mL

B. Use PCB activated with the n-hydroxysuccinimidyl ester (PCB-NHS) at54 mmoles/mL

C; Make BSA solution 33% DMP

Add 446 mL DMF per 10 mg BSA, mix

Vortex while adding 54.4 mL of PCB-NHS per 10 mg BSA (20 fold excess)

D. Incubate 1 hour at room temperature

E. Centrifuge for 5 minutes at 14,000 rpm

F. Put the supernatant through a G-25 column (1.5 mL for each disposable10 mL column), equilibrated with PBS/0.05% azide.

G. Collect protein fraction

IV. Assay

A. Attach an absorbent pad to top and bottom end of strip

B. Add several drops of sample to bottom absorbent pad of the strip andallow to “wick” up the strip

C. Allow sample to wick up entire length of strip

D. If color develops in the detection zone (approximately 1 cm from endof strip), then the sample contains PCB.

While the invention has been described in connection with a preferredembodiment, it is not intended to limit the scope of the invention tothe particular form set forth, but, on the contrary, it is intended tocover such alternatives, modifications and equivalents as may beincluded within the spirit and scope of the invention as defined by theappended claims.

What is claimed is:
 1. A method for detecting the presence and amount ofan analyte in a sample suspected of containing the analyte, the methodemploying a chromatographic strip-positive readout binding assay device,the binding assay device comprising, a chromatographic strip with aproximal end and a distal end having a length and narrow width thatconveys fluids in a fluid flow direction along the length toward thedistal end of the strip, the strip comprising the following zones; asample contact zone where fluid sample suspected of containing ananalyte may be contacted with the strip; a trapping zone located at, ordownstream from, the sample contact zone comprising an immobilizedsecond binding partner that binds to a first binding partner/anti-firstbinding partner complex that is not bound to analyte in the sample; anda detection zone located on the strip downstream from the trapping zonecomprising an immobilized binding moiety specific for the first bindingpartner/anti-first binding partner complex that is not bound to theimmobilized second binding partner, the method comprising, combining asample to be tested with a diffusible labeled anti-first binding partnerand a diffusible unlabeled first binding partner to form a sample-assaycomponent mixture, applying the sample-assay component mixture to thesample contact zone, and detecting the presence of label in thedetection zone, wherein the detection of label in the detection zoneindicates the presence of analyte in the sample.
 2. The method of claim1, wherein the first binding partner is analyte receptor that binds toanalyte in the sample.
 3. The method of claim 1, wherein the firstbinding partner is a conjugate of analyte and carrier and the analytedoes not compete with the first binding partner for binding to theanti-first binding partner.
 4. The method of claim 1, wherein theimmobilized second binding partner is analyte receptor.
 5. The method ofclaim 1, wherein the immobilized second binding partner isanalyte-carrier conjugate.
 6. The method of claim 1, wherein the firstbinding partner is selected from the group consisting of antibodies,aryl hydrocarbon receptors, hormone receptors, nucleic acid hybridizingoligomers, and synthetic binding partners, and the second bindingpartner is selected from the group consisting of antigens, haptens, arylhydrocarbons, hormones, and DNA/RNA fragments.
 7. The method of claim 1,wherein the second binding partner is selected from the group consistingof antibodies, aryl hydrocarbon receptors, hormone receptors, nucleicacid hybridizing oligomers, and synthetic binding partners, and thefirst binding partner is selected from the group consisting of antigens,haptens, aryl hydrocarbons, hormones, and DNA/RNA fragments and theanalyte does not compete with the first binding partner for binding tothe anti-first binding partner.
 8. The method of claim 1, furthercomprising filter material overlaid on the sample contact zone.
 9. Themethod of claim 1, further comprising absorbent material attached to thedistal end of the chromatographic strip.
 10. The method of claim 1,further comprising pretreating the sample prior to applying the sampleto the sample contact zone, wherein the pretreatment extracts,concentrates or releases analyte from the sample.
 11. The method ofclaim 1, wherein the label is selected from the group consisting ofcolored latex beads, gold particles, carbon black, horseradishperoxidase, alkaline phosphatase, bioluminescent photoproteins, andchemiluminescent compounds.
 12. The method of claim 11, furthercomprising measuring the amount of color localized at the detectionzone.
 13. A method for detecting the presence and amount of an analytein a sample suspected of containing the analyte, the method employing achromatographic strip-positive readout binding assay device, the bindingassay device comprising, a chromatographic strip with a proximal end anda distal end having a length and narrow width that conveys fluids in afluid flow direction along the length toward the distal end of thestrip, the strip comprising the following zones; a sample contact zonewhere fluid sample suspected of containing an analyte may be contactedwith the strip; a labeled anti-first binding partner zone disposed at,or downstream from the sample contact zone comprising a diffusiblelabeled anti-first binding partner; a trapping zone located at, ordownstream from, the sample contact zone comprising an immobilizedsecond binding partner that binds to a first binding partner/anti-firstbinding partner complex that is not bound to analyte in the sample; anda detection zone located on the strip downstream from the trapping zonecomprising an immobilized binding moiety specific for the first bindingpartner/anti-first binding partner complex that is not bound to theimmobilized second binding partner, the method comprising, combining asample to be tested with a diffusible unlabeled first binding partner toform a sample-assay component mixture, applying the sample-assaycomponent mixture to the sample contact zone, and detecting the presenceof label in the detection zone, wherein the detection of label in thedetection zone indicates the presence of analyte in the sample.
 14. Amethod for detecting the presence and amount of an analyte in a samplesuspected of containing the analyte, the method employing achromatographic strip-positive readout binding assay device, the bindingassay device comprising, a chromatographic strip with a proximal end anda distal end having a length and narrow width that conveys fluids in afluid flow direction along the length toward the distal end of thestrip, the strip comprising the following zones; a sample contact zonewhere fluid sample suspected of containing an analyte may be contactedwith the strip; a first binding partner zone disposed at, or downstreamfrom the sample contact zone comprising a diffusible unlabeled firstbinding partner that binds to a labeled anti-first binding partner toform a diffusible first binding partner/anti-first binding partnercomplex; a trapping zone located at, or downstream from, the samplecontact zone comprising an immobilized second binding partner that bindsto a first binding partner/anti-first binding partner complex that isnot bound to analyte in the sample; and a detection zone located on thestrip downstream from the trapping zone comprising an immobilizedbinding moiety specific for the first binding partner/anti-first bindingpartner complex that is not bound to the immobilized second bindingpartner, the method comprising, combining a sample to be tested with adiffusible labeled anti-first binding partner to form a sample-assaycomponent mixture, applying the sample-assay component mixture to thesample contact zone, and detecting the presence of label in thedetection zone, wherein the detection of label in the detection zoneindicates the presence of analyte in the sample.